Methods for the identification of inhibitors of CAX1-like Ca+2/H+ antiporter activity in plants

ABSTRACT

The present inventors have discovered that CAX1-like H+/Ca +2  antiporter is essential for plant growth. Specifically, the inhibition of CAX1-like H+/Ca +2  antiporter gene expression in plant seedlings results in reduced growth and abnormal development. Thus, CAX1-like H+/Ca +2  antiporter is useful as a target for the identification of herbicides. Accordingly, the present invention provides methods for the identification of herbicides by measuring the activity of a CAX1-like H+/Ca +2  antiporter in the presence and absence of a compound, where an alteration of CAX1-like H+/Ca +2  antiporter activity in the presence of the compound indicates the compound as a candidate for a herbicide.

FIELD OF THE INVENTION

[0001] The invention relates generally to plant molecular biology. Inparticular, the invention relates to methods for the identification ofherbicides.

BACKGROUND OF THE INVENTION

[0002] The traditional approach to herbicide development is to spraychemicals, produced in milligram or greater quantity, on plants and thento monitor plant growth. While spray and observe approach has resultedin the identification of commercially important herbicides, risingcosts, and efficacy and safety concerns are challenging its futureproductivity. Accordingly, there is a need to identify herbicide targetsso that compound libraries can be screened for herbicidal activity inhigher through-put in vitro or cell-based assays. Inhibitors of theidentified targets can then be selected and confirmed as havingherbicidal activity using conventional assays.

[0003] Calcium plays a central role in signal transduction by eukaryoticcells. A complex mechanism exists to control Ca⁺² in a localizedfashion. Ca⁺² -ATPase, Ca⁺²/H+ antiporter (also referred to as Ca+²/H+exchanger), Ca+²/Na+ antiporter, and Ca+² channel are involved in theregulation of cytosolic Ca⁺² concentration (Yuasa & Maeshima (2000)Plant Physiology 124: 1069-1078). In animals, Ca⁺² is primarilymobilized from the endoplasmic reticulum. In plants and fungi thevacuole is a primary pool of Ca⁺² and the vacuolar membrane has twodistinct active transport systems for Ca⁺²: Ca⁺²-ATPase and Ca⁺²/H+antiporter (Sanders et al. (1999) Plant Cell 11: 691-706; Sze et al.(2000) Annu. Rev. Plant Physiol. Plant Mol. Biol. 51: 433-462).

[0004] Yeast mutants defective in Ca⁺² transport have beencharacterized. Yeast vcx1 pmc1 mutants deleted for the vacuolar H+/Ca⁺²antiporter (VCX1) and vacuolar P-type Ca⁺²-ATPase (PMC1) genes arehypersensitive to Ca⁺² in the growth medium (Cunningham & Fink (1994) J.Cell Biol. 124: 351-363; Cunningham & Fink (1996) Mol. Cell Biol. 16:2226-2237). Three Arabidopsis thaliana genes, CAX1, CAX2, and CAX3, havebeen shown to suppress the yeast vcx1 pmc1 mutant Ca⁺² accumulationdefect (Hirschi et al. (1996) Proc. Natl. Acad. Sci. 93: 8782-8786;Shigaki T. et al. (2001) J. Biol. Chem. 276:43152-9). The Arabidopsisgenes, CAX1 and CAX2, encode high efficiency and low efficiency H+/Ca⁺²antiporters, respectively. The CAX3 gene encodes a CAX1-like H+/Ca⁺²antiporter.

[0005] The present invention discloses CAX1-like H+/Ca⁺² antiporters astargets for the evaluation of plant growth regulators, especiallyherbicide compounds, in plants.

SUMMARY OF THE INVENTION

[0006] The present inventors have discovered that antisense expressionof a CAX1-like H+/Ca⁺² antiporter cDNA (Accession No.: AF256228) inArabidopsis causes reduced growth and abnormal development. Thus, thepresent inventors have discovered that CAX1-like H+/Ca⁺² antiporter isessential for normal plant development and growth, and is useful as atarget for the identification of herbicides.

[0007] Accordingly, in one embodiment the present invention providesmethods for identifying a compound as a candidate for a herbicide,comprising: measuring the activity of a CAX1-like H+/Ca⁺² antiporter inthe presence and absence of a compound, wherein an alteration of theCAX1-like H+/Ca⁺² antiporter activity in the presence of the compoundindicates the compound as a candidate for a herbicide.

[0008] In another embodiment, the present invention provides methods forthe identification of compounds that inhibit CAX1-like H+/Ca⁺²antiporter expression or activity, comprising: measuring the growth in ahigh Ca⁺² medium of a Ca⁺² sensitive pmc1 vcx1 strain of mutant yeastcells in the presence and absence of a compound, wherein the mutantyeast cells express a heterologous CAX1-like H+/Ca⁺² antiporterpolypeptide that rectifies the Ca⁺² sensitive phenotype; measuring thegrowth in a normal Ca⁺² medium of a Ca⁺² sensitive pmc1 vcx1 strain ofmutant yeast cells in the presence and absence of the compound, whereinthe mutant yeast cells express a heterologous CAX1-like H+/Ca⁺²antiporter polypeptide that rectifies the Ca⁺² sensitive phenotype; andcomparing the growth in steps (a) and (b), wherein a decrease in growthin step (a) in the presence relative to the absence of the compound, andno change in growth in step (b) between the presence and absence of thecompound indicates the compound as a candidate for a herbicide.

[0009] In another embodiment, the invention provides methods foridentifying a compound as a candidate for a herbicide, comprising:measuring the expression of a CAX1-like H+/Ca⁺² antiporter in a plant,or tissue thereof, in the presence and absence of a compound; andcomparing the expression of the CAX1-like H+/Ca⁺² antiporter in thepresence and absence of the compound, wherein an altered expression inthe presence of the compound indicates that the compound is a candidatefor a herbicide.

DETAILED DESCRIPTION OF THE INVENTION

[0010] The term “bDNA” refers to branched DNA.

[0011] As used herein, the terms “CAX1-like H+/Ca⁺² antiporter,”“Ca⁺²/H+exchanger” and “Ca⁺²/H+ exchange protein” are interchangeable,and refer to an enzyme that catalyzes vacuolar H+/Ca⁺² antiporteractivity. As used herein, the term “CAX1-like H+/Ca⁺² antiporter” meanseither a nucleic acid encoding a polypeptide or a polypeptide, whereinthe polypeptide has at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity oreach integer unit of sequence identity from 40-100% in ascending orderto either Arabidopsis CAX1-like H+/Ca⁺² antiporter protein (CAX3; SEQ IDNO:2) or Arabidopsis truncated CAX1-like H+/Ca⁺² antiporter protein (SEQID NO:4) and at least 10%, 25%, 50%, 75%, 80%, 90%, 95%, or 99% activityor each integer unit of activity from 10-100% in ascending order of theactivity of Arabidopsis CAX1-like H+/Ca⁺² antiporter protein (CAX3; SEQID NO:2) or Arabidopsis truncated CAX1-like H+/Ca⁺² antiporter protein(SEQ ID NO:4), respectively. Examples of CAX1-like H+/Ca⁺² antiporter'sinclude, but are not limited to, CAX1-like H+/Ca⁺² antiporter from Vignaradiate, CAX1-like H+/Ca⁺² antiporter from Oryza sativa and CAX1-likeH+/Ca⁺² antiporter from Zea Mays.

[0012] By “Ca⁺² sensitive” is meant that growth of a pmc1 vcx1 mutant isinhibited on culture medium containing a high concentration of Ca⁺² butnot one containing a normal concentration of Ca⁺². By “high Ca⁺² medium”is meant a medium containing sufficient Ca⁺² to cause a reduction ingrowth of a pmc1 vcx1 mutant relative to a wild-type yeast strain. It isunderstood by one of ordinary skill in the art that the concentration ofCa⁺² in a high Ca⁺² medium will vary depending on the type andcomposition of growth medium used (e.g. liquid versus solid), the lengthof the growth period measured, and with the particular mutant yeaststrain. Examples of normal concentrations of Ca⁺² for growth of a pmc1vcx1 mutant yeast strain on solid media range from 0 to 100 mM. Examplesof high concentrations of Ca⁺² for growth of a pmc1 vcx1 mutant yeaststrain on solid media range from 200 to 300 mM. Normal concentrations ofCa⁺² for growth of a pmc1 vcx1 mutant yeast strain in liquid media arelower than those for growth on solid media. For example, a highconcentration of Ca⁺² for growth of a pmc1 vcx1 mutant yeast strain inliquid media is 100 mM.

[0013] As used herein, the term “CDNA” means complementarydeoxyribonucleic acid.

[0014] As used herein, the term “ELISA” means enzyme-linkedimmunosorbent assay.

[0015] As used herein, the term “GUS” means β-glucouronidase.

[0016] The term “herbicide,” as used herein, refers to a compound usefulfor killing or suppressing the growth of at least one plant, plant cell,plant tissue or seed.

[0017] The phrase “heterologous CAX1-like H+/Ca⁺² antiporter,” as usedherein, refers to any CAX1-like H+/Ca⁺² antiporter polypeptide that isencoded by a nucleic acid molecule that has been transformed orintroduced into mutant yeast cells being used in a cell based assay foridentifying inhibitors of CAX1-like H+/Ca⁺² antiporter activity.

[0018] The term “inhibitor,” as used herein, refers to a chemicalsubstance that inactivates the enzymatic activity of CAX1-like H+/Ca⁺²antiporter or substantially reduces the level of enzymatic activity,wherein “substantially” means a reduction at least as great as thestandard deviation for a measurement, preferably a reduction by 50%,more preferably a reduction of at least one magnitude, i.e. to 10%. Theinhibitor may function by interacting directly with the enzyme, acofactor of the enzyme, the substrate of the enzyme, or any combinationthereof.

[0019] A polynucleotide is “introduced” into a plant cell by any means,including transfection, transformation or transduction, electroporation,particle bombardment, agroinfection and the like. The introducedpolynucleotide is maintained in the cell stably if it is incorporatedinto a non-chromosomal autonomous replicon or integrated into the plantchromosome. Alternatively, the introduced polynucleotide is present onan extra-chromosomal non-replicating vector and be transiently expressedor transiently active.

[0020] As used herein, the term “PCR” means polymerase chain reaction.

[0021] The “percent (%) sequence identity” between two polynucleotide ortwo polypeptide sequences is determined according to the either theBLAST program (Basic Local Alignment Search Tool, Altschul and Gish(1996) Meth Enzymol 266: 460-480; Altschul (1990) J Mol Biol 215: 403-410) or using Smith Waterman Alignment (Smith and Waterman (1981) AdvAppl Math 2:482) using the default settings and the version current atthe time of filing). It is understood that for the purposes ofdetermining sequence identity when comparing a DNA sequence to an RNAsequence, a thymine nucleotide is equivalent to a uracil nucleotide.

[0022] “Plant” refers to whole plants, plant organs and tissues (e.g.,stems, roots, ovules, stamens, leaves, embryos, meristematic regions,callus tissue, gametophytes, sporophytes, pollen, microspores and thelike) seeds, plant cells and the progeny thereof.

[0023] By “plant CAX1-like H+/Ca⁺² antiporter” is meant a protein foundin at least one plant, and which catalyzes vacuolar H+/Ca⁺² antiporteractivity. The CAX1-like H+/Ca⁺² antiporter is from any plant, includingmonocots, dicots, C3 plants, C4 plants and/or plants that are classifiedas neither C3 nor C4 plants.

[0024] By “polypeptide” is meant a chain of at least four amino acidsjoined by peptide bonds. The chain is linear, branched, circular orcombinations thereof. The polypeptides may contain amino acid analogsand other modifications, including, but not limited to glycosylated orphosphorylated residues.

[0025] The present inventors have discovered that inhibition ofCAX1-like H+/Ca⁺² antiporter gene expression inhibits the growth anddevelopment of plant seedlings. Antisense expression of Arabidopsisthaliana CAX3 cDNA (AF256228; SEQ ID NO:1) resulted in abnormal growthand development. The protein encoded by the Arabidopsis CAX3 gene (SEQID NO:2) has a high degree of homology to Arabidopsis CAX1 high affinityH+/Ca⁺² antiporter (AAB05913). Thus, the inventors are the first todemonstrate that CAX1-like H+/Ca⁺² antiporters are useful targets forthe identification of herbicides.

[0026] Accordingly, the invention provides methods for identifyingcompounds that inhibit CAX1-like H+/Ca⁺² antiporter protein activity.Such methods include binding assays, activity assays and assays forCAX1-like H+/Ca⁺² antiporter gene expression. The compounds identifiedby the methods of the invention are useful as herbicides.

[0027] In one embodiment, the invention provides a method for theidentification of a compound as a herbicide, comprising: measuring theactivity of a CAX1-like H+/Ca⁺² antiporter in the presence and absenceof the compound, wherein an alteration of the CAX1-like H+/Ca⁺²antiporter activity in the presence of the compound indicates thecompound as a candidate for a herbicide.

[0028] By “CAX1-like H+/Ca⁺² antiporter” is meant an enzyme thatcatalyzes vacuolar H+/Ca⁺² antiporter activity. In one embodiment of theinvention, the CAX1-like H+/Ca⁺² antiporter has the amino acid sequenceof a naturally occurring CAX1-like H+/Ca⁺² antiporter found in a plant,animal or microorganism. In another embodiment of the invention, theCAX1-like H+/Ca⁺² antiporter has an amino acid sequence derived from anaturally occurring sequence. In another embodiment the CAX1-likeH+/Ca⁺² antiporter is a plant CAX1-like H+/Ca⁺² antiporter.

[0029] One example of a cDNA encoding anArabidopsis CAX1-like H+/Ca⁺²antiporter is set forth in SEQ ID NO:1 (TIGR database locus At3g51860;CAX3). The CAX1-like H+/Ca⁺² antiporter polypeptide encoded by SEQ IDNO:1 is set forth in SEQ ID NO:2. A nucleic acid molecule encoding anN-terminal 55 amino acid truncated and C-terminal 6-His tag peptidefusion of Arabidopsis CAX1-like H+/Ca⁺² antiporter is set forth in SEQID NO:3. The fusion polypeptide encoded by SEQ ID NO:3 is set forth inSEQ ID NO:4. Other examples of CAX1-like H+/Ca⁺² antiporters include theVigna radiata CAX1-like H+/Ca⁺² antiporter protein set forth in SEQ IDNO:5 (Accession No. BAA25753). Another example of a CAX1-like H+/Ca⁺²antiporter is a Oryza sativa CAX1-like H+/Ca⁺² antiporter protein setforth in SEQ ID NO:6 (Accession No. BAB89095). Another example of aCAX1-like H+/Ca⁺² antiporter is a Zea Mays CAX1-like H+/Ca⁺² antiporterprotein set forth in SEQ ID NO:7 (Accession No. AAF91350). Vignaradiate, Oryza sativa and Zea Mays CAX1-like H+/Ca⁺² antiporters thathave N-terminal amino acid truncations similar to that of SEQ ID NO:4are also useful in the methods of the invention.

[0030] In one embodiment, the CAX1-like H+/Ca⁺² antiporter is anArabidopsis CAX1-like H+/Ca⁺² antiporter. Arabidopsis species include,but are not limited to, Arabidopsis arenosa, Arabidopsis bursifolia,Arabidopsis cebennensis, Arabidopsis croatica, Arabidopsis griffithiana,Arabidopsis halleri, Arabidopsis himalaica, Arabidopsis korshinskyi,Arabidopsis lyrata, Arabidopsis neglecta, Arabidopsis pumila,Arabidopsis suecica, Arabidopsis thaliana and Arabidopsis wallichii.

[0031] In various embodiments, the CAX1-like H+/Ca⁺² antiporter can befrom barnyard grass (Echinochloa crus-galli), crabgrass (Digitariasanguinalis), green foxtail (Setana viridis), perennial ryegrass (Loliumperenne), hairy beggarticks (Bidens pilosa), nightshade (Solanumnigrum), smartweed (Polygonum lapathifolium), velvetleaf (Abutilontheophrasti), common lambsquarters (Chenopodium album L.), Brachiaraplantaginea, Cassia occidentalis, Ipomoea aristolochiaefolia, Ipomoeapurpurea, Euphorbia heterophylla, Setaria spp, Amaranthus retroflexus,Sida spinosa, Xanthium strumarium and the like.

[0032] CAX1-like H+/Ca⁺² antiporter polypeptides having at least 40%sequence identity with either Arabidopsis CAX1-like H+/Ca⁺² antiporterprotein (CAX3; SEQ ID NO:2) or Arabidopsis truncated CAX1-like H+/Ca⁺²antiporter protein (SEQ ID NO:4) protein are also useful in the methodsof the invention. In one embodiment, the sequence identity is at least40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 99%, or any integer from 40-100% sequence identity inascending order with either Arabidopsis CAX1-like H+/Ca⁺² antiporterprotein (CAX3; SEQ ID NO:2) or Arabidopsis truncated CAX1-like H+/Ca⁺²antiporter protein (SEQ ID NO:4). In addition, it is preferred thatCAX1-like H+/Ca⁺² antiporter polypeptides of the invention have at least10% of the activity of either Arabidopsis CAX1-like H+/Ca⁺² antiporterprotein (CAX3; SEQ ID NO:2) or Arabidopsis truncated CAX1-like H+/Ca⁺²antiporter protein (SEQ ID NO:4). CAX1-like H+/Ca 2 antiporterpolypeptides of the invention have at least 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or at least 90% ofthe activity of Arabidopsis CAX1-like H+/Ca⁺² antiporter protein (SEQ IDNO:2) or Arabidopsis truncated CAX1-like H+/Ca⁺² antiporter protein (SEQID NO:4).

[0033] Polypeptides consisting essentially of SEQ ID NO:4 are alsouseful in the methods of the invention. For the purposes of the presentinvention, a polypeptide consisting essentially of SEQ ID NO:4 has atleast 90% sequence identity with Arabidopsis CAX1-like H+/Ca⁺²antiporter fusion protein (SEQ ID NO:4) and at least 10% of the activityof SEQ ID NO:4. A polypeptide consisting essentially of SEQ ID NO:4 hasat least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, or 99% sequenceidentity with SEQ ID NO:4 and at least 25%, 50%, 75%, or 90% of theactivity of Arabidopsis CAX1-like H+/Ca⁺² antiporter (SEQ ID NO:4).

[0034] Examples of polypeptides consisting essentially of SEQ ID NO:4include, but are not limited to, polypeptides having the amino acidsequence of SEQ ID NO:4 with the exception that one or more of the aminoacids are substituted with structurally similar amino acids providing a“conservative amino acid substitution.” Conservative amino acidsubstitutions are well known to those of skill in the art. Particularexamples of polypeptides consisting essentially of SEQ ID NO:4 includepolypeptides having 1, 2, or 3 conservative amino acid substitutionsrelative to SEQ ID NO:4.

[0035] Other examples of polypeptides consisting essentially of SEQ IDNO:4 include polypeptides having the sequence of SEQ ID NO:4, but withtruncations at either or both the 3′ and the 5′ end. For example,polypeptides consisting essentially of SEQ ID NO:4 include polypeptideshaving 1, 2, or 3 amino acids residues removed from either or both 3′and 5′ ends relative to SEQ ID NO:4. Additional examples of polypeptidesconsisting essentially of SEQ ID NO:4 include polypeptides having 1, 2,3, 4, 5, 6, 7, 8, 9, or 10 fewer amino acids residues truncated from theN-terminus relative to SEQ ID NO:4. In addition, CAX1-like H+/Ca⁺²antiporter polypeptides consisting essentially of SEQ ID NO:4 can befusion proteins, such as SEQ ID NO:4 but having other fused polypeptidesor amino acid sequences to aid in secretion and/or isolation as is knownto those of skill in the art.

[0036] Fragments of a CAX1-like H+/Ca⁺² antiporter polypeptide areuseful in the methods of the invention. In one embodiment, the CAX1-likeH+/Ca⁺² antiporter fragments include an intact or nearly intact epitopethat occurs on the biologically active wild-type CAX1-like H+/Ca⁺²antiporter. For example, the fragments comprise at least 10 consecutiveamino acids of CAX1-like H+/Ca⁺² antiporter of SEQ ID NO:2. Thefragments comprise at least 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90,100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425 orat least 458 consecutive amino acid residues of CAX1-like H+/Ca⁺²antiporter of SEQ ID NO:2. Polypeptides comprising at least 50 aminoacids having at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99%sequence identity with at least 50 consecutive amino acid residues ofSEQ ID NO:2 are also useful in the methods of the invention. In oneembodiment, the fragment is from an Arabidopsis CAX1-like H+/Ca⁺²antiporter. In one embodiment, the fragment contains an amino acidsequence conserved among plant CAX1-like H+/Ca⁺² antiporter sequences.

[0037] Thus, in another embodiment, the invention provides a method foridentifying a compound as a candidate for a herbicide, comprising:contacting a compound with a CAX1-like H+/Ca⁺² antiporter polypeptideselected from the group consisting of: a polypeptide set forth in SEQ IDNO:2 or SEQ ID NO:4; a CAX1-like H+/Ca⁺² antiporter polypeptideconsisting essentially of SEQ ID NO:4; a polypeptide comprising at least10 consecutive amino acids of SEQ ID NO:2; a CAX1-like H+/Ca⁺²antiporter polypeptide having at least 50% sequence identity with SEQ IDNO:2 or SEQ ID NO:4; and a CAX1-like H+/Ca⁺² antiporter polypeptidecomprising at least 50 amino acids having at least 50% sequence identitywith at least 50 consecutive amino acid residues of SEQ ID NO:2 or SEQID NO:4; and detecting the presence and/or absence of binding betweenthe compound and the polypeptide, wherein binding indicates that thecompound is a candidate for a herbicide.

[0038] Any technique for detecting the binding of a ligand to its targetis useful in the methods of the invention. For example, the ligand andtarget are combined in a buffer. Many methods for detecting the bindingof a ligand to its target are known in the art, and include, but are notlimited to the detection of an immobilized ligand-target complex or thedetection of a change in the properties of a target when it is bound toa ligand. For example, in one embodiment, an array of immobilizedcandidate ligands is provided. The immobilized ligands are contactedwith a CAX1-like H+/Ca⁺² antiporter protein or a fragment or variantthereof, the unbound protein is removed and the bound CAX1-like H+/Ca⁺²antiporter is detected. In a preferred embodiment, bound CAX1-likeH+/Ca⁺² antiporter is detected using a labeled binding partner, such asa labeled antibody. In a variation of this assay, CAX1-like H+/Ca ⁺²antiporter is labeled prior to contacting the immobilized candidateligands. Preferred labels include fluorescent or radioactive moieties.In other embodiments of the invention, detection methods includefluorescence correlation spectroscopy (FCS) and FCS-related confocalnanofluorimetric methods.

[0039] In another embodiment of the invention, compounds are tested ascandidate herbicides based on ability to inhibit CAX1-like H+/Ca⁺²antiporter enzyme activity. The compounds are tested using either invitro or cell based enzyme assays. Alternatively, compounds are testedby direct application to a plant or plant cell, or expressing ittherein, and monitoring the plant or plant cell for changes or decreasesin growth, development, viability or alterations in gene expression.

[0040] A decrease in growth occurs where the herbicide candidate causesat least a 10% decrease in the growth of the plant or plant cells, ascompared to the growth of the plants or plant cells in the absence ofthe herbicide candidate. A decrease in viability occurs where at least20% of the plants cells, or portions of the plant contacted with theherbicide candidate, are nonviable. Preferably, the growth or viabilitywill be decreased by at least 40%. More preferably, the growth orviability will be decreased by at least 50%, 75%, or at least 90% ormore. Methods for measuring plant growth and cell viability are known tothose skilled in the art. It is possible that a candidate compound mayhave herbicidal activity only for certain plants or certain plantspecies.

[0041] The ability of a compound to inhibit CAX1-like H+/Ca⁺² antiporteractivity can be detected using cell based assays in which a CAX1-likeH+/Ca⁺² antiporter polypeptide complements the Ca⁺² sensitivity of ayeast pmc1 vcx1 double mutant. Growth of the pmc1 vcx1 mutant isinhibited on culture medium containing a high concentration of Ca⁺².Expression of a heterologous CAX1-like H+/Ca⁺² antiporter proteinrelieves the inhibition and enables the mutant to grow in the presenceof a high Ca⁺² concentration. CAX1-like H+/Ca⁺² antiporter inhibitorsare identified by their ability to confer Ca⁺² sensitive growth on theCAX1-like H+/Ca⁺² antiporter-expressing pmc1 vcx1 mutant.

[0042] Thus, in one embodiment, the invention provides methods for theidentification of compounds that inhibit CAX1-like H+/Ca⁺² antiporteractivity, comprising: measuring the growth in a high Ca⁺² medium of aCa⁺² sensitive pmc1 vcx1 strain of mutant yeast cells in the presenceand absence of a compound, wherein the mutant yeast cells express aheterologous CAX1-like H+/Ca⁺² antiporter polypeptide that rectifies theCa⁺² sensitive phenotype; measuring the growth in a normal Ca⁺² mediumof a Ca⁺² sensitive pmc1 vcx1 strain of mutant yeast cells in thepresence and absence of the compound, wherein the mutant yeast cellsexpress a heterologous CAX1-like H+/Ca ⁺² antiporter polypeptide thatrectifies the Ca⁺² sensitive phenotype; and comparing the growth insteps (a) and (b), wherein a decrease in growth in step (a) in thepresence relative to the absence of the compound, and no change ingrowth in step (b) between the presence and absence of the compoundindicates the compound as a candidate for a herbicide. The phrase“heterologous CAX1-like H+/Ca⁺² antiporter” is herein intended to meanany CAX1-like H+/Ca⁺² antiporter polypeptide that is encoded by anucleic acid molecule that has been transformed or introduced into themutant yeast cells.

[0043] In one embodiment of the invention, the CAX1-like H+/Ca⁺²antiporter is the polypeptide set forth in SEQ ID NO:2. In anotherembodiment, the CAX1-like H+/Ca⁺² antiporter is the polypeptide setforth in SEQ ID NO:4. In another embodiment, the CAX1-like H+/Ca⁺²antiporter is a polypeptide consisting essentially of SEQ ID NO:4. Inanother embodiment, the CAX1-like H+/Ca⁺² antiporter is an ArabidopsisCAX1-like H+/Ca⁺² antiporter polypeptide. In another embodiment, theCAX1-like H+/Ca⁺² antiporter is a plant CAX1-like H+/Ca⁺² antiporter. Inanother embodiment the CAX1-like H+/Ca⁺² antiporter is Vigna radiata CAX1-like H+/Ca⁺² antiporter set forth in SEQ ID NO:5. In anotherembodiment the CAX1-like H+/Ca⁺² antiporter is Oryza sativa CAX1-likeH+/Ca⁺² antiporter set forth in SEQ ID NO:6. In another embodiment theCAX1-like H+/Ca⁺² antiporter is Zea Mays CAX1-like H+/Ca⁺² antiporterset forth in SEQ ID NO:7.

[0044] Enzymatically active fragments of Arabidopsis CAX1-like H+/Ca⁺²antiporter set forth in SEQ ID NO:2 or SEQ ID NO:4 are also useful inthe methods of the invention. For example, an enzymatically activepolypeptide comprising at least 50 consecutive amino acid residues andat least 10% of the activity of Arabidopsis CAX1-like H+/Ca⁺² antiporterset forth in SEQ ID NO:2 or SEQ ID NO:4 are useful in the methods of theinvention. The fragments comprise at least 15, 20, 25, 30, 35, 40, 50,60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350,375, 400, 425 or at least 458 consecutive amino acid residues ofCAX1-like H+/Ca⁺² antiporter of SEQ ID NO:2 or SEQ ID NO:4. In addition,enzymatically active fragments of CAX1-like H+/Ca⁺² antiporter's usefulin the methods of the invention include polypeptides comprising at least50 amino acids having at least 10% of the activity of SEQ ID NO:2 or SEQID NO:4 and at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99%sequence identity with at 50 consecutive amino acid residues of SEQ IDNO:2 or SEQ ID NO:4, respectively, are useful in the methods of theinvention. Most preferably, the enzymatically active polypeptidecomprises at least 50 amino acids, has at least 50% sequence identitywith at least 50 consecutive amino acid residues of SEQ ID NO:2 or SEQID NO:4 and at least 25%, 75% or at least 90% of the activity thereof.

[0045] Thus, in other embodiments of the invention, the CAX1-likeH+/Ca⁺² antiporter is a polypeptide selected from the group consistingof: a polypeptide having at least 50% sequence identity with ArabidopsisCAX1-like H+/Ca⁺² antiporter set forth in SEQ ID NO:2 or SEQ ID NO:4 andhaving at least 10% of the activity thereof; a polypeptide comprising atleast 50 consecutive amino acids ofArabidopsis CAX1-like H+/Ca⁺²antiporter set forth in SEQ ID NO:2 or SEQ ID NO:4 and having at least10% of the activity thereof; and a polypeptide comprising at least 50amino acids, having at least 50% sequence identity with at least 50consecutive amino acids of Arabidopsis CAX1-like H+/Ca⁺² antiporter setforth in SEQ ID NO:2 or SEQ ID NO:4 and having at least 10% of theactivity thereof.

[0046] As an alternative to cell-based assays, the invention alsoprovides plant based assays. In one embodiment, the invention provides amethod for identifying a compound as a candidate for a herbicide,comprising: a) measuring the expression or activity of a CAX1-likeH+/Ca⁺² antiporter in a plant, or tissue thereof, in the absence of acompound; b) measuring the expression or activity of the CAX1-likeH+/Ca⁺² antiporter in the plant, or tissue thereof, in the presence ofthe compound; and c) comparing the expression or activity of theCAX1-like H+/Ca⁺² antiporter in steps (a) and (b), wherein an alteredexpression or activity in the presence of the compound indicates thatthe compound is a candidate for a herbicide. In one embodiment, theplant or tissue thereof is Arabidopsis thaliana.

[0047] In the methods of the invention, expression of a CAX1-likeH+/Ca⁺² antiporter in a plant, or tissue thereof, is measured bydetecting the CAX1-like H+/Ca⁺² antiporter primary transcript or mRNA,CAX1-like H+/Ca⁺² antiporter polypeptide or CAX1-like H+/Ca⁺² antiporterenzymatic activity. Methods for detecting the expression of RNA andproteins are known to those skilled in the art. (See, for example,Current Protocols in Molecular Biology, Ausubel et al., eds., GreenePublishing and Wiley-Interscience, New York, 1995). However, the methodof detection is not critical to the invention. Methods for detectingCAX1-like H+/Ca⁺² antiporter RNA include, but are not limited to,amplification assays such as quantitative PCR, and/or hybridizationassays such as Northern analysis, dot blots, slot blots, in-situhybridization, transcriptional fusions using a CAX1-like H+/Ca⁺²antiporter promoter fused to a reporter gene, bDNA assays, andmicroarray assays.

[0048] Methods for detecting protein expression include, but are notlimited to, immunodetection methods such as Western blots, His Tag andELISA assays, polyacrylamide gel electrophoresis, mass spectroscopy, andenzymatic assays. Also, any reporter gene system is useful to detectCAX1-like H+/Ca⁺² antiporter protein expression. For detection usinggene reporter systems, a polynucleotide encoding a reporter protein isfused in frame with CAX1-like H+/Ca⁺² antiporter, so as to produce achimeric polypeptide. Methods for using reporter systems are known tothose skilled in the art. Examples of reporter genes include, but arenot limited to, chloramphenicol acetyltransferase (Gorman et al. (1982)Mol Cell Biol 2: 1104; Prost et al. (1986) Gene 45: 107-111),β-galactosidase (Nolan et al. (1988) Proc Natl Acad Sci USA 85:2603-2607), alkaline phosphatase (Berger et al. (1988) Gene 66: 10),luciferase (De Wet et al. (1987) Mol Cell Biol 7: 725-737),β-glucuronidase (GUS), fluorescent proteins, chromogenic proteins andthe like.

[0049] Chemicals, compounds, or compositions identified by the abovemethods as modulators of CAX1-like H+/Ca⁺² antiporter expression oractivity are useful for controlling plant growth. For example, compoundsthat inhibit plant growth are applied to a plant or expressed in a plantto prevent plant growth. Thus, the invention provides a method forinhibiting plant growth, comprising contacting a plant with a compoundidentified by the methods of the invention as having herbicidalactivity.

[0050] Herbicides and herbicide candidates identified by the methods ofthe invention are useful for controlling the growth of undesired plants,including including monocots, dicots, C3 plants, C4 plants, and plantsthat are neither C3 nor C4 plants. Examples of undesired plants include,but are not limited, to barnyard grass (Echinochloa crus-galli),crabgrass (Digitaria sanguinalis), green foxtail (Setana viridis),perennial ryegrass (Lolium perenne), hairy beggarticks (Bidens pilosa),nightshade (Solanum nigrum), smartweed (Polygonum lapathifolium),velvetleaf (Abutilon theophrasti), common lambsquarters (Chenopodiumalbum L.), Brachiara plantaginea, Cassia occidentalis, Ipomoeaaristolochiaefolia, Ipomoea purpurea, Euphorbia heterophylla, Setariaspp, Amaranthus retroflexus, Sida spinosa, Xanthium strumarium and thelike.

EXPERIMENTAL

[0051] Plant Growth Conditions

[0052] Unless, otherwise indicated, all plants were grown in ScottsMetro-Mix™ soil (the Scotts Company) or a similar soil mixture in anenvironmental growth room at 22° C., 65% humidity, 65% humidity and alight intensity of ˜100 μ-E m⁻² s⁻¹ supplied over 16 hour day period.

[0053] Seed Sterilization

[0054] All seeds were surface sterilized before sowing onto phytagelplates using the following protocol.

[0055] 1. Place approximately 20-30 seeds into a labeled 1.5 ml conicalscrew cap tube. Perform all remaining steps in a sterile hood usingsterile technique.

[0056] 2. Fill each tube with 1 ml 70% ethanol and place on rotisseriefor 5 minutes.

[0057] 3. Carefully remove ethanol from each tube using a sterileplastic dropper; avoid removing any seeds.

[0058] 4. Fill each tube with 1 ml of 30% Clorox and 0.5% SDS solutionand place on rotisserie for 10 minutes.

[0059] 5. Carefully remove bleach/SDS solution.

[0060] 6. Fill each tube with 1 ml sterile DI H₂O; seeds should bestirred up by pipetting of water into tube. Carefully remove water.Repeat 3 to 5 times to ensure removal of Clorox/SDS solution.

[0061] 7. Fill each tube with enough sterile dI H₂O for seed plating(˜200-400 μl). Cap tube until ready to begin seed plating.

[0062] Plate Growth Assays

[0063] Surface sterilized seeds were sown onto plate containing 40 mlhalf strength sterile MS (Murashige and Skoog, no sucrose) medium and 1%Phytagel using the following protocol:

[0064] 1. Using pipette man and 200 μl tip, carefully fill tip with seedsolution. Place 10 seeds across the top of the plate, about 1/4 inchdown from the top edge of the plate.

[0065] 2. Place plate lid ¾ of the way over the plate and allow to dryfor 10 minutes.

[0066] 3. Using sterile micropore tape, seal the edge of the plate wherethe top and bottom meet.

[0067] 4. Place plates stored in a vertical rack in the dark at 4° C.for three days.

[0068] 5. Three days after sowing, the plates transferred into a growthchamber with a day and night temperature of 22 and 20° C., respectively,65% humidity and a light intensity of ˜100 μ-E m⁻² s⁻¹ supplied over 16hour day period.

[0069] 6. Beginning on day 3, daily measurements are carried out totrack the seedlings development until day 14. Seedlings are harvested onday 14 (or when root length reaches 6 cm) for root and rosette analysis.

EXAMPLE 1 Construction of a Transgenic Plant expressing the Driver

[0070] The “Driver” is an artificial transcription factor comprising achimera of the DNA-binding domain of the yeast GAL4 protein (amino acidresidues 1-147) fused to two tandem activation domains of herpes simplexvirus protein VP16 (amino acid residues 413-490). Schwechheimer et al.(1998) Plant Mol Biol 36:195-204. This chimeric driver is atranscriptional activator specific for promoters having GAL4 bindingsites. Expression of the driver is controlled by two tandem copies ofthe constitutive CaMV 35S promoter.

[0071] The driver expression cassette was introduced into Arabidopsisthaliana by agroinfection. Transgenic plants that stably expressed thedriver transcription factor were obtained.

EXAMPLE 2 Construction of CAX1-Like H+/Ca⁺² Antiporter AntisenseExpression Cassettes in a Binary Vector

[0072] A fragment of the Arabidopsis thaliana cDNA corresponding to SEQID NO:1 was ligated into the PacI/AscI sites of an E. coli/Agrobacteriumbinary vector in the antisense orientation to yield an antisenseexpression cassette and a constitutive chemical resistance expressioncassette located between right and left T-DNA borders. In thisconstruct, transcription of the antisense RNA is controlled by anartificial promoter active only in the presence of the drivertranscription factor described above. The artificial promoter containsfour contiguous binding sites for the GAL4 transcriptional activatorupstream of a minimal promoter comprising a TATA box. The ligated DNAwas transformed into E. coli. Kanamycin resistant clones were selectedand purified. DNA was isolated from each clone and characterized by PCRand sequence analysis confirming the presence of the antisenseexpression cassette.

EXAMPLE 3 Transformation of Agrobacterium with the CAX1-Like H+/Ca⁺²Antiporter Antisense Expression Cassette

[0073] The binary vector described in Example 2 was transformed intoAgrobacterium tumefaciens by electroporation. Transformed Agrobacteriumcolonies were isolated using chemical selection. DNA was prepared frompurified resistant colonies and the inserts were amplified by PCR andsequenced to confirm sequence and orientation.

EXAMPLE 4 Construction of Arabidopsis CAX1-Like H+/Ca⁺² AntiporterAntisense Target Plants

[0074] The CAX1-like H+/Ca⁺² antiporter antisense expression cassettewas introduced into Arabidopsis thaliana wild-type plants by thefollowing method. Five days prior to agroinfection, the primaryinflorescence of Arabidopsis thaliana plants grown in 2.5 inch pots wereclipped to enhance the emergence of secondary bolts.

[0075] At two days prior to agroinfection, 5 ml LB broth (10 g/LPeptone, 5 g/L Yeast extract, 5 g/L NaCl, pH 7.0 plus 25 mg/L kanamycinadded prior to use) was inoculated with a clonal glycerol stock ofAgrobacterium carrying the desired DNA. The cultures were incubatedovernight at 28° C. at 250 rpm until the cells reached stationary phase.The following morning, 200 ml LB in a 500 ml flask was inoculated with500 μl of the overnight culture and the cells were grown to stationaryphase by overnight incubation at 28° C. at 250 rpm. The cells werepelleted by centrifugation at 8000 rpm for 5 minutes. The supernatantwas removed and excess media was removed by setting the centrifugebottles upside down on a paper towel for several minutes. The cells werethen resuspended in 500 ml infiltration medium (autoclaved 5% sucrose)and 250 μl/L Silwet L-77™ (84% polyalkyleneoxide modifiedheptamethyltrisiloxane and 16% allyloxypolyethyleneglycol methyl ether),and transferred to a one liter beaker.

[0076] The previously clipped Arabidopsis plants were dipped into theAgrobacterium suspension so that all above ground parts were immersedand agitated gently for 10 seconds. The dipped plants were then coveredwith a tall clear plastic dome to maintain the humidity, and returned tothe growth room. The following day, the dome was removed and the plantswere grown under normal light conditions until mature seeds wereproduced. Mature seeds were collected and stored desiccated at 4° C.

[0077] Transgenic Arabidopsis T1 seedlings were selected. Approximately70 mg seeds from an agrotransformed plant were mixed approximately 4:1with sand and placed in a 2 ml screw cap cryo vial. One vial of seedswas then sown in a cell of an 8 cell flat. The flat was covered with adome, stored at 4° C. for 3 days, and then transferred to a growth room.The domes were removed when the seedlings first emerged. After theemergence of the first primary leaves, the flat was sprayed uniformlywith a herbicide corresponding to the chemical resistance marker plus0.005% Silwet (50 μl/L) until the leaves were completely wetted. Thespraying was repeated for the following two days.

[0078] Ten days after the first spraying resistant plants weretransplanted to 2.5 inch round pots containing moistened sterile pottingsoil. The transplants were then sprayed with herbicide and returned tothe growth room. The herbicide resistant plants represented stablytransformed T1 plants.

EXAMPLE 5 Effect of CAX1-Like H+/Ca⁺² Antiporter Antisense Expression inArabidopsis Seedlings

[0079] The T1 CAX1-like H+/Ca⁺² antiporter antisense target plants fromthe transformed plant lines obtained in Example 4 were crossed with theArabidopsis transgenic driver line described above. The resulting F1seeds were then subjected to a plate assay to observe seedling growthover a 2-week period. Seedlings were inspected for growth anddevelopment. Antisense expression of the CAX1-like H+/Ca⁺² antiportergene in three separate lines resulted in significantly impaired growthand abnormal development, indicating that the CAX1-like H+/Ca⁺²antiporter gene is an essential gene for normal plant growth anddevelopment. Six of eight plants from the first transgenic line, threeof six plants from the second transgenic line, and five of seven plantsfrom the third transgenic line showed reduced growth and/or abnormaldevelopment. Thus, each of the three transgenic lines containing theantisense construct for CAX1-like Ca⁺²/H+ antiporter exhibitedsignificant seedling abnormalities.

EXAMPLE 6 Construction of a Ca⁺² Sensitive pmc1 vcx1 Mutant Yeast Strain

[0080]Saccharomyces cerevisiae having a double mutation in both PMC1 andVCX1 genes (pmc1 vcx1 double mutants) exhibits Ca⁺² Sensitivity(Cunningham & Fink (1994) J. Cell Biol. 124: 351-363; Cunningham & Fink(1996) Mol. Cell Biol. 16: 2226-2237). PMC1 encodes a vacuolarCa⁺²-transporting ATPase. PMC1 is a member of the cation transportingP-type ATPase superfamily and functions to pump Ca⁺² out of thecytoplasm into the vacuole. VCX1 is a calcium transport (H+/Ca⁺²exchange) protein of the vacuolar membrane. VCX1 transports H+ out ofthe vacuole into the cytoplasm and Ca⁺² out of the cytoplasm into thevacuole. By “Ca⁺² sensitive” is meant that growth of a pmc1 vcx1 mutantis inhibited on culture medium containing a high concentration of Ca⁺²but not one containing a normal concentration of Ca^(+2.)

[0081] A pmc1Δ::KanMX mutant strain was purchased from Open Biosystems(cat. no. YSC1021-548753) and designated as PGY1. The entire PMC1gene-coding region of strain PGY1 was replaced with a KanMX selectablemarker, conferring resistance to the antibiotic geneticin (G418). Inaddition, PGY1 is a histidine auxotroph as a result of a his3Δ1mutation. A vcx1 mutation was introduced into PGY1 using homologousrecombination to replace the entire VCX1 coding region with the wildtype HIS3 gene. Putative pmc1Δ::KanMX vcx1Δ::HIS3 double mutants wereselected by their ability to grow on medium lacking histidine. Thepresence of both the pmc1 and vcx1 mutations was verified by PCR for twoseparate strains.

EXAMPLE 7 Complementation of the Ca⁺² Sensitive pmc1 vcx1 Mutant YeastStrain by Expression of a Heterologous CAX1-Like H+/Ca⁺² AntiporterProtein

[0082] It has been demonstrated that the Ca⁺² sensitivity of yeast pmc1vcx1 mutants can be complemented by Arabidopsis calcium exchangers CAX1,CAX2 and CAX3 (Hirsehi K. et al. (1996) Proc. Nat. Acad. Sci. 93:8782-6;Shigaki T. et al. (2001) J. Biol. Chem. 276:43152-9) and by theToxoplasma gondii Ca⁺²-transporting ATPase TgA1 (Luo S. et al. (2001)EMBO J 20:55-64). Expression of CAX protein relieves the Ca⁺² inhibitionand enables the mutant to grow in the presence of a high Ca⁺²concentration. The two pmc1Δ vcx1Δ strains from Example 6 were used totest for complementation by a heterologous CAX1-like H+/Ca⁺² antiportergene.

[0083] The two PCR-verified mutant strains were transformed with yeastexpression vectors expressing full-length (SEQ ID NO:1) and truncatedforms (SEQ ID NO:3) of a CAX1-like H+/Ca⁺² antiporter to test whetherthe CAX1-like H+/Ca⁺² antiporter genes complemented the Ca⁺²-sensitivity of the pmc1 vcx1 mutants. As a control, the double mutantwas transformed with empty vector. The Ca⁺² sensitivity of the strainswas examined by spotting serial dilutions onto Ca⁺² containingraffinose-galactose plates that support maximal induction of theCAX1-like H+/Ca⁺² antiporter genes. Under these conditions all of theyeast strains grew equivalently at Ca⁺² concentrations between 0 and 100mM. At Ca⁺² concentrations between 200 and 300 mM, pmc1 vcx1 mutantsthat expressed the truncated CAX1-like H+/Ca⁺² antiporter protein (SEQID NO:4) demonstrated more robust growth than the non-expressing mutantsor the mutants expressing the full-length CAX1-like H+/Ca⁺² antiporterprotein (SEQ ID NO:2).

[0084] The results are consistent with previous studies showing that theN-terminus of the Arabidopsis calcium exchangers contain anautoregulatory region that must be removed for the proteins to becomefunctional in yeast (Luo S. et al. (2001) EMBO J 20:55-64). For furtheranalysis, the vector control strain was designated, PGY15, and thetruncated CAX1-like H+/Ca⁺² antiporter protein (SEQ ID NO:4)-expressingstrain was designated, PGY17. RT-PCR was used to demonstrate thatCAX1-like H+/Ca⁺² antiporter expression occurred in yeast strain PGY17only upon induction with galactose. For this experiment, the controlstrain, PGY15, and the CAX1-like H+/Ca⁺² antiporter-expressing strain,PGY17, were cultured in growth media containing either glucose orraffinose and galactose. Glucose is the favored carbon source for yeast.When yeast is grown on glucose-containing media the expression of genesthat are required for galactose metabolism is repressed. When yeast isgrown on galactose or raffinose-galactose-containing media that lacksglucose, the galactose metabolic network is induced.

[0085] In strain PGY17, CAX1-like H+/Ca⁺² antiporter is under control ofthe GAL1-10 promoter and, thus, CAX1-like H+/Ca⁺² antiporter expressionshould only be observed when this strain is grown ongalactose-containing medium. This is consistent with the observed RT-PCRresults. CAX1-like H+/Ca⁺² antiporter expression was only observed inPGY17 grown in galactose-containing medium but not glucose-containingmedium. As expected, no CAX1-like H+/Ca⁺² antiporter expression wasobserved in the control strain, PGY15, grown on either glucose orgalactose-containing medium.

EXAMPLE 8 Assay for the Identification of Inhibitors of CAX1-LikeH+/Ca⁺² Antiporter Activity

[0086] CAX1-like H+/Ca⁺² antiporter inhibitors are identified by thedisappearance of CAX1-like H+/Ca⁺² antiporter complementation of growthof a CAX1-like H+/Ca⁺² antiporter-expressing pmc1 vcx1 mutant yeaststrain grown on high Ca⁺² media. The identified inhibitors have noeffect on growth of the CAX1-like H+/Ca⁺² antiporter-expressing pmc1vcx1 mutant yeast strain grown on normal Ca⁺² media.

[0087] While the foregoing describes certain embodiments of theinvention, it will be understood by those skilled in the art thatvariations and modifications may still fall within the scope of theinvention.

1 7 1 1380 DNA Arabidopsis thaliana 1 atgggaagta tcgtggagcc atgggcagcaatcgccgaga acggaaacgc aaacgtgacc 60 gcgaaaggct cgagcaggga gctgcgacatgggagaacag cacacaacat gtcttcatcg 120 tcgctaagga agaaatcaga cctgagattggttcagaaag ttccatgcaa aactctcaag 180 aacattctct ctaatcttca agaagtcattcttggtacta agcttactct cttatttctc 240 gccatccctc tcgccattct tgccaattcttacaactacg gtcgtccgtt gatatttgga 300 ctgagcttga tagggctgac acctctagctgagcgagtta gctttttgac agagcaacta 360 gctttctaca ctggtccaac agtgggcggtttgttgaacg cgacttgtgg aaacgcgaca 420 gagctgataa tcgcgatact agcgttggccaataacaaag tggcagtggt gaaatactct 480 ctattgggtt caattctctc aaaccttctcttggttcttg gcacttccct cttctttggt 540 ggtatcgcca atatccgccg cgagcagcggttcgaccgga aacaagccga tgtgaacttc 600 ttcttgctgc ttatgggcct gttgtgtcatttgctgccat tattgttaaa atatgcagca 660 accggcgaag tatcgacctc tatgattaacaaaatgtcgc tcactctgtc gcggacaagc 720 agcatagtta tgcttattgc ttacattgcttatctcatct tccagctctg gactcaccgc 780 caattgtttg aggcacaaca ggatgatgatgatgcatatg atgatgaggt tagtgttgaa 840 gaaactccag tgataggatt ctggagcggatttgcttggc tcgttgggat gacaatagtc 900 atcgcattgc tatcagagta tgttgtggacacgatcgagg atgcatcgga ctcatgggga 960 ctatcagtga gtttcataag catcatattgcttcccattg ttgggaatgc ggctgagcat 1020 gctggagcca tcattttcgc attcaagaacaagctcgaca tatctctagg ggttgcgttg 1080 ggctctgcaa ctcagatttc tttgttcgtggtcccattga gtgttatcgt tgcgtggatc 1140 ctgggaataa aaatggatct caactttaacatccttgaaa ctagctctct agctttggcc 1200 attatcatca cagccttcac tttacaggatggaacttctc attacatgaa gggactggtt 1260 ctattgttat gctatgtcat catcgcggcgtgtttcttcg tcgaccaaat tccccaacca 1320 aatgatttgg acgtgggact tcaacccatgaacaatttgg gagaagtttt ctcagcttaa 1380 2 459 PRT Arabidopsis thaliana 2Met Gly Ser Ile Val Glu Pro Trp Ala Ala Ile Ala Glu Asn Gly Asn 1 5 1015 Ala Asn Val Thr Ala Lys Gly Ser Ser Arg Glu Leu Arg His Gly Arg 20 2530 Thr Ala His Asn Met Ser Ser Ser Ser Leu Arg Lys Lys Ser Asp Leu 35 4045 Arg Leu Val Gln Lys Val Pro Cys Lys Thr Leu Lys Asn Ile Leu Ser 50 5560 Asn Leu Gln Glu Val Ile Leu Gly Thr Lys Leu Thr Leu Leu Phe Leu 65 7075 80 Ala Ile Pro Leu Ala Ile Leu Ala Asn Ser Tyr Asn Tyr Gly Arg Pro 8590 95 Leu Ile Phe Gly Leu Ser Leu Ile Gly Leu Thr Pro Leu Ala Glu Arg100 105 110 Val Ser Phe Leu Thr Glu Gln Leu Ala Phe Tyr Thr Gly Pro ThrVal 115 120 125 Gly Gly Leu Leu Asn Ala Thr Cys Gly Asn Ala Thr Glu LeuIle Ile 130 135 140 Ala Ile Leu Ala Leu Ala Asn Asn Lys Val Ala Val ValLys Tyr Ser 145 150 155 160 Leu Leu Gly Ser Ile Leu Ser Asn Leu Leu LeuVal Leu Gly Thr Ser 165 170 175 Leu Phe Phe Gly Gly Ile Ala Asn Ile ArgArg Glu Gln Arg Phe Asp 180 185 190 Arg Lys Gln Ala Asp Val Asn Phe PheLeu Leu Leu Met Gly Leu Leu 195 200 205 Cys His Leu Leu Pro Leu Leu LeuLys Tyr Ala Ala Thr Gly Glu Val 210 215 220 Ser Thr Ser Met Ile Asn LysMet Ser Leu Thr Leu Ser Arg Thr Ser 225 230 235 240 Ser Ile Val Met LeuIle Ala Tyr Ile Ala Tyr Leu Ile Phe Gln Leu 245 250 255 Trp Thr His ArgGln Leu Phe Glu Ala Gln Gln Asp Asp Asp Asp Ala 260 265 270 Tyr Asp AspGlu Val Ser Val Glu Glu Thr Pro Val Ile Gly Phe Trp 275 280 285 Ser GlyPhe Ala Trp Leu Val Gly Met Thr Ile Val Ile Ala Leu Leu 290 295 300 SerGlu Tyr Val Val Asp Thr Ile Glu Asp Ala Ser Asp Ser Trp Gly 305 310 315320 Leu Ser Val Ser Phe Ile Ser Ile Ile Leu Leu Pro Ile Val Gly Asn 325330 335 Ala Ala Glu His Ala Gly Ala Ile Ile Phe Ala Phe Lys Asn Lys Leu340 345 350 Asp Ile Ser Leu Gly Val Ala Leu Gly Ser Ala Thr Gln Ile SerLeu 355 360 365 Phe Val Val Pro Leu Ser Val Ile Val Ala Trp Ile Leu GlyIle Lys 370 375 380 Met Asp Leu Asn Phe Asn Ile Leu Glu Thr Ser Ser LeuAla Leu Ala 385 390 395 400 Ile Ile Ile Thr Ala Phe Thr Leu Gln Asp GlyThr Ser His Tyr Met 405 410 415 Lys Gly Leu Val Leu Leu Leu Cys Tyr ValIle Ile Ala Ala Cys Phe 420 425 430 Phe Val Asp Gln Ile Pro Gln Pro AsnAsp Leu Asp Val Gly Leu Gln 435 440 445 Pro Met Asn Asn Leu Gly Glu ValPhe Ser Ala 450 455 3 1236 DNA Arabidopsis thaliana 3 atgtgcaaaactctcaagaa cattctctct aatcttcaag aagtcattct tggtactaag 60 cttactctcttatttctcgc catccctctc gccattcttg ccaattctta caactacggt 120 cgtccgttgatatttggact gagcttgata gggctgacac ctctagctga gcgagttagc 180 tttttgacagagcaactagc tttctacact ggtccaacag tgggcggttt gttgaacgcg 240 acttgtggaaacgcgacaga gctgataatc gcgatactag cgttggccaa taacaaagtg 300 gcagtggtgaaatactctct attgggttca attctctcaa accttctctt ggttcttggc 360 acttccctcttctttggtgg tatcgccaat atccgccgcg agcagcggtt cgaccggaaa 420 caagccgatgtgaacttctt cttgctgctt atgggcctgt tgtgtcattt gctgccatta 480 ttgttaaaatatgcagcaac cggcgaagta tcgacctcta tgattaacaa aatgtcgctc 540 actctgtcgcggacaagcag catagttatg cttattgctt acattgctta tctcatcttc 600 cagctctggactcaccgcca attgtttgag gcacaacagg atgatgatga tgcatatgat 660 gatgaggttagtgttgaaga aactccagtg ataggattct ggagcggatt tgcttggctc 720 gttgggatgacaatagtcat cgcattgcta tcagagtatg ttgtggacac gatcgaggat 780 gcatcggactcatggggact atcagtgagt ttcataagca tcatattgct tcccattgtt 840 gggaatgcggctgagcatgc tggagccatc attttcgcat tcaagaacaa gctcgacata 900 tctctaggggttgcgttggg ctctgcaact cagatttctt tgttcgtggt cccattgagt 960 gttatcgttgcgtggatcct gggaataaaa atggatctca actttaacat ccttgaaact 1020 agctctctagctttggccat tatcatcaca gccttcactt tacaggatgg aacttctcat 1080 tacatgaagggactggttct attgttatgc tatgtcatca tcgcggcgtg tttcttcgtc 1140 gaccaaattccccaaccaaa tgatttggac gtgggacttc aacccatgaa caatttggga 1200 gaagttttctcagctcatca tcatcatcat cattaa 1236 4 411 PRT Arabidopsis thaliana 4 MetCys Lys Thr Leu Lys Asn Ile Leu Ser Asn Leu Gln Glu Val Ile 1 5 10 15Leu Gly Thr Lys Leu Thr Leu Leu Phe Leu Ala Ile Pro Leu Ala Ile 20 25 30Leu Ala Asn Ser Tyr Asn Tyr Gly Arg Pro Leu Ile Phe Gly Leu Ser 35 40 45Leu Ile Gly Leu Thr Pro Leu Ala Glu Arg Val Ser Phe Leu Thr Glu 50 55 60Gln Leu Ala Phe Tyr Thr Gly Pro Thr Val Gly Gly Leu Leu Asn Ala 65 70 7580 Thr Cys Gly Asn Ala Thr Glu Leu Ile Ile Ala Ile Leu Ala Leu Ala 85 9095 Asn Asn Lys Val Ala Val Val Lys Tyr Ser Leu Leu Gly Ser Ile Leu 100105 110 Ser Asn Leu Leu Leu Val Leu Gly Thr Ser Leu Phe Phe Gly Gly Ile115 120 125 Ala Asn Ile Arg Arg Glu Gln Arg Phe Asp Arg Lys Gln Ala AspVal 130 135 140 Asn Phe Phe Leu Leu Leu Met Gly Leu Leu Cys His Leu LeuPro Leu 145 150 155 160 Leu Leu Lys Tyr Ala Ala Thr Gly Glu Val Ser ThrSer Met Ile Asn 165 170 175 Lys Met Ser Leu Thr Leu Ser Arg Thr Ser SerIle Val Met Leu Ile 180 185 190 Ala Tyr Ile Ala Tyr Leu Ile Phe Gln LeuTrp Thr His Arg Gln Leu 195 200 205 Phe Glu Ala Gln Gln Asp Asp Asp AspAla Tyr Asp Asp Glu Val Ser 210 215 220 Val Glu Glu Thr Pro Val Ile GlyPhe Trp Ser Gly Phe Ala Trp Leu 225 230 235 240 Val Gly Met Thr Ile ValIle Ala Leu Leu Ser Glu Tyr Val Val Asp 245 250 255 Thr Ile Glu Asp AlaSer Asp Ser Trp Gly Leu Ser Val Ser Phe Ile 260 265 270 Ser Ile Ile LeuLeu Pro Ile Val Gly Asn Ala Ala Glu His Ala Gly 275 280 285 Ala Ile IlePhe Ala Phe Lys Asn Lys Leu Asp Ile Ser Leu Gly Val 290 295 300 Ala LeuGly Ser Ala Thr Gln Ile Ser Leu Phe Val Val Pro Leu Ser 305 310 315 320Val Ile Val Ala Trp Ile Leu Gly Ile Lys Met Asp Leu Asn Phe Asn 325 330335 Ile Leu Glu Thr Ser Ser Leu Ala Leu Ala Ile Ile Ile Thr Ala Phe 340345 350 Thr Leu Gln Asp Gly Thr Ser His Tyr Met Lys Gly Leu Val Leu Leu355 360 365 Leu Cys Tyr Val Ile Ile Ala Ala Cys Phe Phe Val Asp Gln IlePro 370 375 380 Gln Pro Asn Asp Leu Asp Val Gly Leu Gln Pro Met Asn AsnLeu Gly 385 390 395 400 Glu Val Phe Ser Ala His His His His His His 405410 5 444 PRT Vigna radiata 5 Met Gly Ser His Gln His Glu Pro Trp LeuLeu Glu Asn Gly Asn Pro 1 5 10 15 Lys Val Leu Thr Arg Glu Met Arg HisGly Arg Thr Ala His Ser Lys 20 25 30 Ser Ser Asn Ser Leu Arg Thr Lys SerAsp Arg Thr Leu Val Ser Lys 35 40 45 Val Pro Cys Ala Thr Ile Arg Asn ValLeu Phe Asn Leu Gln Glu Val 50 55 60 Ile Leu Gly Thr Lys Leu Ser Ile LeuIle Pro Ala Ile Pro Val Ala 65 70 75 80 Ile Val Ala Glu Tyr Cys Gly PheGly Arg Pro Trp Val Phe Val Leu 85 90 95 Ser Leu Leu Gly Leu Thr Pro LeuAla Glu Arg Val Ser Phe Ile Thr 100 105 110 Glu Gln Val Ala Phe Tyr ThrGly Pro Thr Val Gly Gly Leu Leu Asn 115 120 125 Ala Thr Cys Gly Asn ValThr Glu Leu Ile Ile Ala Ile Phe Ala Leu 130 135 140 Ser Ser Asn Lys IleAla Val Val Lys Tyr Ser Leu Leu Gly Ser Ile 145 150 155 160 Leu Ser AsnLeu Leu Leu Val Leu Gly Thr Ser Leu Leu Cys Gly Gly 165 170 175 Ile AlaAsn Val Gly Leu Glu Gln Lys Tyr Asp Arg Arg Gln Gly Asp 180 185 190 ValAsn Ser Leu Met Leu Leu Leu Ala Leu Leu Cys Tyr Leu Leu Pro 195 200 205Met Leu Phe Lys Tyr Ser Ala Ala Ser Ala Ala Leu Thr Val Asp Pro 210 215220 Ser Leu His Leu Ser Arg Ala Ser Ser Ile Val Met Leu Ile Ala Tyr 225230 235 240 Val Val Tyr Ile Ile Phe Gln Leu Trp Thr His Arg Gln Leu PheGlu 245 250 255 Ala Glu Asp Glu Asp Glu Asp Asp Asn Asn Gly Ser Asp GluGln Ala 260 265 270 Val Ile Gly Leu Trp Ser Gly Ile Ala Trp Leu Ile GlyMet Thr Val 275 280 285 Phe Ile Ala Leu Leu Ser Glu Tyr Val Val Asp ThrIle Glu Asp Ala 290 295 300 Ser Asp Ser Trp Gly Leu Ser Val Ser Phe LeuSer Ile Ile Leu Leu 305 310 315 320 Pro Ile Val Gly Asn Ala Ala Glu HisAla Gly Ala Val Ile Phe Ala 325 330 335 Phe Lys Asn Lys Leu Asp Ile SerLeu Gly Val Ala Leu Gly Ser Ala 340 345 350 Thr Gln Ile Ala Met Phe ValVal Pro Leu Cys Val Ile Val Ala Trp 355 360 365 Thr Met Gly Val Lys MetAsp Leu Asn Phe Asn Ile Leu Glu Thr Gly 370 375 380 Ser Val Ala Leu AlaIle Ile Val Thr Ser Phe Thr Leu Gln Asp Gly 385 390 395 400 Thr Ser HisTyr Met Lys Gly Leu Val Leu Leu Leu Cys Tyr Ile Val 405 410 415 Ile GlyAla Cys Phe Phe Val Gln Arg Thr Pro Phe Asn Gln Ala Asp 420 425 430 ValThr Asn Val Ile Pro Asn Gly Val Leu Ser Ala 435 440 6 424 PRT Oryzasativa 6 Met Ser Ser Ser Ser Leu Arg Lys Lys Ser Asp Ala Ala Leu Val Arg1 5 10 15 Lys Val Pro Val Ala Pro Leu Arg Pro Leu Leu Ala Asn Leu GlnGlu 20 25 30 Val Phe Leu Ala Thr Lys Leu Ala Val Leu Phe Pro Ala Val ProLeu 35 40 45 Ala Ile Ala Ala Gln Cys Phe Arg Phe Asp Gln Val Trp Val PheAla 50 55 60 Leu Ser Leu Leu Gly Leu Ile Pro Leu Ala Glu Arg Val Ser PheLeu 65 70 75 80 Thr Glu Gln Ile Ala Leu Tyr Thr Gly Pro Thr Val Gly GlyLeu Leu 85 90 95 Asn Ala Thr Cys Gly Asn Ala Thr Glu Leu Ile Ile Ala LeuPhe Ala 100 105 110 Leu Leu Lys Gly Lys Ile Glu Val Val Lys Cys Ser LeuLeu Gly Ser 115 120 125 Val Leu Ser Asn Leu Leu Leu Val Leu Gly Thr SerLeu Phe Cys Gly 130 135 140 Gly Val Val Asn Leu Gly Ala Arg Gln Pro TyrAsp Arg Asn Gln Ser 145 150 155 160 Asp Val Ser Thr Ala Leu Leu Phe LeuAla Val Leu Cys His Ser Ala 165 170 175 Pro Leu Leu Leu Arg Tyr Ala ValAla Ala Gly Glu His Ser Val Ser 180 185 190 Ala Thr Ser Ala Ala Ala SerLeu Asp Leu Ser Arg Ala Cys Ser Phe 195 200 205 Val Met Leu Ala Ser TyrVal Ala Tyr Leu Phe Phe Gln Leu Lys Thr 210 215 220 His Arg Gln Leu PheGlu Pro Gln Glu Val Asp Gly Gly Asp Ala Gly 225 230 235 240 Asp Asp AspGlu Glu Pro Ala Leu Gly Phe Ala Ser Ala Leu Phe Trp 245 250 255 Leu AlaLeu Met Thr Ala Val Ile Ser Val Leu Ser Glu Tyr Val Val 260 265 270 GlyThr Ile Glu Pro Thr Ser Gln Ser Trp Gly Leu Ser Val Ser Phe 275 280 285Ile Ser Ile Ile Leu Leu Pro Ile Val Gly Asn Ala Ala Glu His Ala 290 295300 Gly Ala Ile Ile Phe Ala Leu Lys Asn Lys Leu Asp Ile Thr Leu Gly 305310 315 320 Val Ala Leu Gly Ser Ala Thr Gln Ile Ser Met Phe Val Val ProLeu 325 330 335 Ser Val Leu Val Ala Trp Ile Met Gly Val Gln Met Asp LeuAsp Phe 340 345 350 Lys Leu Leu Glu Thr Gly Ser Leu Phe Met Ala Val LeuVal Thr Ala 355 360 365 Phe Thr Leu Gln Asp Gly Thr Ser His Tyr Leu LysGly Ile Leu Leu 370 375 380 Leu Leu Cys Tyr Ile Val Ile Gly Ala Cys PhePhe Val Ala Arg Gln 385 390 395 400 Pro Ala Gly His Ala Asn Ser Asn GlyAla Leu Leu Asp Val Pro Thr 405 410 415 Gly Ser Met Ser Val Gln Ala Ala420 7 418 PRT Zea Mays 7 Met His Ala Ser Gly Lys Lys Ser Asp Leu Ala LeuLeu Arg Lys Val 1 5 10 15 Pro Cys Ala Pro Leu Arg Arg Leu Leu Asp AsnLeu Gln Glu Val Leu 20 25 30 Leu Ala Thr Lys Leu Ala Leu Leu Phe Pro AlaVal Leu Leu Ala Ile 35 40 45 Ala Ala Arg Ile Phe His Phe Gly Gln Glu TrpVal Phe Val Leu Ser 50 55 60 Leu Ile Gly Leu Val Pro Leu Ala Glu Arg LeuSer Phe Leu Thr Glu 65 70 75 80 Gln Val Ala Phe Tyr Ile Gly Pro Thr ValGly Gly Leu Leu Asn Ala 85 90 95 Thr Phe Gly Asn Val Thr Glu Val Ile IleAla Ile Phe Ala Leu Tyr 100 105 110 Gln Gly Lys Val Val Val Val Lys CysSer Leu Leu Gly Ser Val Leu 115 120 125 Ser Asn Leu Leu Leu Val Leu GlyThr Ser Leu Phe Phe Gly Gly Leu 130 135 140 Ala Asn Leu Gly Thr Glu GlnLeu Tyr Asp Lys Met Gln Val Asp Val 145 150 155 160 Asn Thr Gly Leu LeuIle Leu Gly Val Leu Cys His Ser Leu Pro Leu 165 170 175 Met Leu Arg TyrAla Val Ser Ser Gly Glu His Ala Glu Ser Ser Trp 180 185 190 Asp Ser GlyLeu Glu Leu Ser Arg Ala Cys Ser Ile Val Met Leu Leu 195 200 205 Ala TyrVal Ala Tyr Leu Phe Phe Gln Leu Lys Thr His Arg Gln Leu 210 215 220 PheGlu Pro Gln Pro Gln Glu Val Glu Asp Asp Gly Asp Asp Ser Val 225 230 235240 Ser Gln Asp Glu Ala Val Leu Gly Phe Ser Ser Ala Met Ile Trp Leu 245250 255 Gly Val Met Thr Leu Met Thr Ala Leu Leu Ser Glu Phe Val Val Ser260 265 270 Thr Ile Glu Ala Ala Ser Glu Ser Trp Glu Leu Ser Val Ser PheIle 275 280 285 Ser Val Ile Leu Ile Pro Ile Val Gly Asn Ala Ala Glu HisAla Gly 290 295 300 Ala Val Ile Phe Ala Phe Lys Asn Asn Leu Asp Ile ThrLeu Gly Val 305 310 315 320 Ser Leu Gly Ser Ala Thr Gln Ile Ser Met PheVal Val Pro Leu Ser 325 330 335 Val Leu Val Ala Trp Ile Met Gly Val ProMet Asp Leu Asp Phe Asn 340 345 350 Leu Leu Glu Thr Gly Cys Leu Phe LeuAla Ile Leu Val Thr Ala Phe 355 360 365 Thr Leu Gln Asp Gly Ser Ser HisTyr Leu Lys Gly Leu Leu Leu Val 370 375 380 Phe Cys Tyr Ile Val Ile SerLeu Cys Phe Phe Val Leu Arg Gln His 385 390 395 400 Gly Asn Gly Ser AsnAsp Asp Gln Val Gly Val Ala Ser Lys Pro Trp 405 410 415 Arg Ile

What is claimed is:
 1. A method for identifying a compound as acandidate for a herbicide, comprising: a) measuring the growth in a highCa⁺² medium of a Ca⁺² sensitive pmc1 vex1 strain of mutant yeast cellsin the presence and absence of a compound, wherein the mutant yeastcells express a heterologous CAX1-like H+/Ca⁺² antiporter polypeptidethat rectifies the Ca⁺² sensitive phenotype; b) measuring the growth ina normal Ca⁺² medium of a Ca⁺² sensitive pmc1 vex1 strain of mutantyeast cells in the presence and absence of the compound, wherein themutant yeast cells express a heterologous CAX1-like H+/Ca⁺² antiporterpolypeptide that rectifies the Ca⁺² sensitive phenotype; and c)comparing the growth in steps (a) and (b), wherein a decrease in growthin step (a) in the presence relative to the absence of the compound, andno change in growth in step (b) between the presence and absence of thecompound indicates the compound as a candidate for a herbicide.
 2. Themethod of claim 1, wherein the CAX1-like H+/Ca⁺² antiporter polypeptideis a plant CAX1-like H+/Ca⁺² antiporter polypeptide.
 3. The method ofclaim 2, wherein the plant is a dicot.
 4. The method of claim 2, whereinthe plant is a monocot.
 5. The method of claim 2, wherein the plant isother than a C3 plant.
 6. The method of claim 2, wherein the plant isother than a C4 plant.
 7. The method of claim 1, wherein the CAX1-likeH+/Ca⁺² antiporter polypeptide is an Arabidopsis CAX1-like H+/Ca⁺²antiporter polypeptide.
 8. The method of claim 1, wherein the CAX1-likeH+/Ca⁺² antiporter polypeptide is SEQ ID NO:4.
 9. The method of claim 1,wherein the CAX1-like H+/Ca⁺² antiporter polypeptide is a polypeptideconsisting essentially of SEQ ID NO:4.
 10. The method of claim 1,wherein the CAX1-like H+/Ca⁺² antiporter polypeptide is a polypeptideselected from the group consisting of: a) a polypeptide having at least50% sequence identity with SEQ ID NO:4 and at least 10% of the activityof SEQ ID NO:4; b) a polypeptide comprising at least 50 consecutiveamino acids of SEQ ID NO:4 and having at least 10% of the activity ofSEQ ID NO:4; and c) a polypeptide comprising at least 50 amino acidshaving at least 50% sequence identity with at least 50 consecutive aminoacids of SEQ ID NO:4 and having at least 10% of the activity of SEQ IDNO:4.
 11. A method for identifying a compound as a candidate for aherbicide, comprising: a) measuring the activity of a CAX1-like H+/Ca⁺²antiporter in the presence and absence of a compound, wherein analteration of the CAX1-like H+/Ca⁺² antiporter activity in the presenceof the compound indicates the compound as a candidate for a herbicide.12. The method of claim 11, wherein the CAX1-like H+/Ca⁺² antiporter isa plant CAX1-like H+/Ca⁺² antiporter.
 13. The method of claim 12,wherein the plant is a dicot.
 14. The method of claim 12, wherein theplant is a monocot.
 15. The method of claim 12, wherein the plant isother than a C3 plant.
 16. The method of claim 12, wherein the plant isother than a C4 plant.
 17. The method of claim 11, wherein the CAX1-likeH+/Ca⁺² antiporter is an Arabidopsis CAX1-like H+/Ca⁺² antiporter. 18.The method of claim 11, wherein the CAX1-like H+/Ca⁺² antiporter is SEQID NO:4.
 19. The method of claim 11, wherein the CAX1-like H+/Ca⁺²antiporter consists essentially of SEQ ID NO:4.
 20. The method of claim11, wherein the CAX1-like H+/Ca⁺² antiporter is a polypeptide selectedfrom the group consisting of: a) a polypeptide having at least 50%sequence identity with SEQ ID NO:4 and at least 10% of the activity ofSEQ ID NO:4; b) a polypeptide comprising at least 50 consecutive aminoacids of SEQ ID NO:4 and having at least 10% of the activity of SEQ IDNO:4; and c) a polypeptide comprising at least 50 amino acids having atleast 50% sequence identity with at least 50 consecutive amino acids ofSEQ ID NO:4 and having at least 10% of the activity of SEQ ID NO:4. 21.A method for identifying a compound as a candidate for a herbicide,comprising: a) measuring the expression of a CAX1-like H+/Ca⁺²antiporter in a plant, or tissue thereof, in the presence and absence ofa compound; and b) comparing the expression of the CAX1-like H+/Ca⁺²antiporter in the presence and absence of the compound, wherein analtered expression in the presence of the compound indicates that thecompound is a candidate for a herbicide.
 22. The method of claim 21,wherein the plant is Arabidopsis.
 23. The method of claim 21, whereinthe expression of the CAX1-like H+/Ca⁺² antiporter is measured bydetecting the CAX1-like H+/Ca⁺² antiporter mRNA.
 24. The method of claim21, wherein the expression of the CAX1-like H+/Ca⁺² antiporter ismeasured by detecting the CAX1-like H+/Ca⁺² antiporter polypeptide. 25.The method of claim 21, wherein the expression of the CAX1-like H+/Ca⁺²antiporter is measured by detecting the CAX1-like H+/Ca⁺² antiporterpolypeptide enzyme activity.
 26. An isolated nucleic acid comprising anucleotide sequence that encodes the polypeptide of SEQ ID NO:4.
 27. Anisolated nucleic acid comprising a nucleotide sequence that encodes apolypeptide consisting essentially of SEQ ID NO:4.
 28. A recombinantpolypeptide consisting essentially of the amino acid sequence of SEQ IDNO:4.
 29. A recombinant polypeptide comprising the amino acid sequenceof SEQ ID NO:4.